Cell culture propagation apparatus

ABSTRACT

Apparatus and method for propagating tissue culture cells. A plurality of spaced-apart, parallel flat plates are assembled. The cells are grown between the plates. Gases or liquid media may be introduced between the plates.

This is a continuation, of application Ser. No. 742,250, filed Nov. 16,1976 and now abandoned.

INTRODUCTION

Recently, it has been recommended that live viral vaccines for human use(poliomyelitis, rubella or measles) be produced by normal human diploidcell strains in cultures. To accomplish this purpose, development of asuitable technique and apparatus for cultivating these cells is neededand desired.

Heretofore, essentially two methods have been available for cellcultivation: one, a monolayer culture technique and the other, asuspension culture technique to which human diploid cells are not yetadaptable. At present, these cells grow only under monolayer cultureconditions.

Conventional monolayer cultivation is performed with glass or plasticbottles or with Petri dishes. Sterilized culture media containing asuspension of cells is placed into the bottles or dishes, the cellsadhere to the bottom surfaces of the vessels, grow, and finally formsheets on the surfaces. The space above the culture media is, of course,filled with air.

Cell cultivation by the above method requires cell growth on thesurfaces of a great many small vessels in order to obtain sufficientamounts for production of biological products. Since cell culturesrequire aseptic handling, treatment of a large number of small vesselsis both cumbersome and time-consuming. Also, the volume occupied bylarge numbers of these vessels is wasteful of space.

Other apparatus and methods recently devised to eliminate the abovedisadvantages, such as the roller bottle cultivation; multi-disc tissuepropagators; multi-plate, and sterilic-type apparatus, while providing alarge surface area in a small, totally-occupied volume which can behandled easily, require movement between the cell sheets and the culturemedia, unlike the static monolayer culture technique, in order toprovide sufficient oxygen supply to the cells due to the larger surfacearea. One means of supplying oxygen utilized by these techniques is tocirculate the oxygenated media, and the other means is the reciprocatingmovement of a cell sheet across the air phase and the liquid phase.

Movement of the cell sheet and/or the culture medium causes difficultyin the maintenance of the microenvironment of the cells, andmicroexudate, for example carpet protein which is secreted from thecells may diffuse, thus inhibiting cell growth. Cells of low density areparticularly affected by loss of the microexudate.

SUMMARY OF THE INVENTION

The present invention relates to a method and apparatus for growinganimal cells, and in particular, to a method and apparatus for growingcell cultures on a large scale. Such large scale growth systems aredesirable in view of the large quantities of cells required in thedevelopment and production of biological products such as viralvaccines, interferon, enzymes such as urokinase, and hormones.

Therefore, it is a principal object of the present invention to providea large surface area for cell growth in a small total volume wherein asufficient oxygen supply is provided without movement of the cells andculture media and thus eliminating any diffusion of microexudate.

This and other objects of the present invention will become more readilyapparent from the following description of the drawings, taken inconjunction with the appended specification.

DRAWINGS

FIG. 1 is a side view of one form of the apparatus of the presentinvention, showing a stacked-dish culture apparatus with the medium andgas being independent of one another;

FIG. 2 is a view, in cross-section, of the apparatus of FIG. 1, in whichthe dishes have overflow passages through which liquids or gases canpass;

FIG. 3 is a side view, in cross-section, of the apparatus as shown inFIG. 2, illustrating diagrammatically one method of distributing thecell suspension in the culture medium;

FIG. 4 is a side view, in cross-section, of the apparatus shown in FIG.2, showing the dishes with two vertical holes having two lateral groovesconnected thereto, through which liquid or gas can pass from one dish toanother;

FIG. 5 is a diagrammatic representation showing the method forinoculating the cell suspension to the culture apparatus as shown inFIG. 4;

FIG. 6 is a view of another form of the apparatus of the presentinvention;

FIG. 7 is a side view of the container culture apparatus of FIG. 6;

FIG. 7a is a side view of the apparatus of FIG. 7, with the lifted edgesof the cell attachment plates standing in a vertical position;

FIG. 7b is a side view of the apparatus of FIG. 7, with the lifted edgesof the cell attachment plate sitting in a horizontal position;

FIG. 7c is a side view of the apparatus of FIG. 7, with the apparatus inan upright position and the cell attachment plates having the cellsuspension on them; and

FIG. 8 is a view of the apparatus of FIG. 7, with overflow passages, andshowing that the cell attachment plates can be removed.

DETAILED DESCRIPTION OF THE INVENTION

Functionally, the present invention is similar to the bottle systemwherein stacks of gas phase, liquid phase and cell layers are repeatedin one apparatus. To employ this system, applicants have shown in thedrawings two types of apparatus, both of which comprise a plurality offlat, spaced-apart plates for cell attachment on which the gas, liquidand cells can exist in parallel position.

Referring now to the drawings, and with particular reference to FIG. 1,culture dishes are provided comprising cell attachment plates 1 on whichcells are grown, and frames 2 enclosing the plates. Inlet/outlet ports 3are provided on the sides of the culture dishes for passage of themedium and gas. These inlet/outlet ports 3 can be stoppered by means ofa screw cap, a rubber stopper or by connection of a stopcock, forexample. The culture dishes are placed between a cover plate 4 and abase plate 5 and are pressed tightly in place by clamps 6. Resilientgaskets 7 are provided to stop any leakage between the uppermost dishand cover plate 4, and/or between dishes. Base plate 5 is provided tosecure the bottom of the lowermost dish but is not necessary when thebottom of the lowermost dish is sufficiently rigid. Attached to the baseplate 5 are adjustable feet 8 by which the cell attachment plate can bekept horizontally as shown in FIG. 2. Overflow passages 9 (FIG. 2) areprovided through which the media, cell suspensions and gases can pass.

In FIG. 3 of the drawings, a method is illustrated for dispensing aliquid, such as a cell suspension, evenly to all dishes. The quantity ofmedia dispensed reaches its maximum when the cell attachment plate ishorizontal and as the angle between the plate and the horizontal levelinclines, the quantity of liquid involved in each dish decreases.

Referring now to FIGS. 4 and 5, inter-dish passages are provided bymeans of two vertical holes 10 connected with two lateral grooves 11leading to the inside of each of the dishes through frames 2, throughwhich the culture media and cells in reservoir 12 (FIG. 5) are pouredfrom the inlet port 13 on cover plate 4 into all the dishes with evendistribution. The height of reservoir 12 (FIG. 5) should be maintainedabove that of the culture apparatus to avoid flowback of the media intothe reservoir.

Thus, by employing the aforementioned form of apparatus and method, aseries of operations such as inoculation of cells, discarding of oldmedia and replenishment of new media can be carried out simply andaseptically in a single action in the multimonolayer culture vessels. Inaddition, aseptically-filtered gases can be introduced through the port13 on the cover or base plate into all the dishes through vertical holes10 and lateral grooves 11 while the apparatus is in a horizontalarrangement.

Although the frames and cell attachment plates employed in thisinvention do not have to be joined together, the frames must have twosealing devices on the upside and downside in order to maintaintightness. Therefore, disposable cell attachment plates can be used whenframes and cell attachment plates are not joined.

Referring now to FIGS. 6 and 7, in this form of the apparatus of thepresent invention, a portion of the edge of the cell attachment plates 1fuses with the container side walls with the remaining portion of theedge of the cell attachment plates being lifted up, thus holding theculture medium on the cell attachment plate. In this form of theapparatus of the present invention, the apparatus is turned laterally sothat all lifted edges of the cell attachment plates stand in a verticalposition as shown in FIG. 7a; the cell suspension is poured through theupper port and the apparatus is again turned laterally so that thelifted edges of the cell attachment plates lie in a horizontal positionas shown in FIG. 7b. In this way, the cell suspension is equallydispensed into the sectionized spaces. Finally, the apparatus is againturned laterally bringing it to an upright position so that the cellsuspension is maintained on the cell attachment plates as shown in FIG.7c. Gases, such as a mixture of CO₂ and air, may be introduced throughthe inlet port to adjust the pH of the culture media, with the containerbeing tightly closed by means of the front panel which has a resilientgasket inside of it.

Culture media and/or cell suspensions can be dispensed evenly throughoverflow passages 20, similar to those of FIG. 3, as illustrated in FIG.8, and as shown in FIG. 8, these dishes with overflow passages can beremoved from the container.

There is no limitation as to size or shape of the cell attachment platesutilized by the present invention, though substantial flatness isrequired. The surface material of the plate should be nontoxic for celladherence and growth, and easily sterilizable, particularly by heating.Since microscopic observation or examination with the naked eye isdesirable on occasion, the cell attachment plates should be transparent.Glass, polyethylene terephthalate, polycarbonate or surface-activatedmethylpentene polymers are suitable substances for the cell attachmentplate. If heat sterilization is not employed, polystyrene can also beused. In the use of methylpentene polymer which is transparent toultraviolet light, sterilization can be carried out by externalradiation with ultraviolet light (germicidal lamp).

Referring to the stacked-dish form of the present invention, the heightof the frame enclosing the plate is the sum of the thickness of theliquid and gas phases and is generally about 4˜50 m/m, but since a largesurface area per total volume is generally required for large scaleanimal cell propagation, the height of the frame may be minimized towithin the range of about 4 to 20 m/m. If the frames are composed ofresilient material such as rubber, additional packings beyond theresilient gaskets provided (FIG. 1) are not necessary. The frames ofthis invention should be heat sterilizable and also non-toxic to thecells, and therefore may be made of stainless steel, aluminum,polycarbonate, methylpentene polymer, rubbers including silicon rubber,glass or other complexed material. If ethylene oxide gas is used as thesterilizing agent, most synthetic polymers can be employed in theculture apparatus. However, ethylene oxide gas is easily absorbed on thepolymers and removal of the gas from the polymers is verytime-consuming. The cover and base plates of the stacked-dish form ofthe present invention should also be transparent, heat sterilizable whenpossible, and non-toxic.

In the container form of the present invention, both the container andfront panel are also preferably made of transparent, heat-sterilizableand non-toxic materials.

The following examples will further illustrate the apparatus and methodof the present invention.

EXAMPLE 1

Human diploid strain WI-38 cells (3×10⁷) grown in five Roux bottles witha cell attachment area of 150 cm² per bottle were trypsinized with 0.25%trypsin and suspended in a nutrient medium comprising:

Eagle's basal medium--699 mls.

7.5% Sodium bicarbonate--13 mls.

3.0% Glutamine--8 mls.

Calf serum--80 mls.

The cell suspension was poured into the apparatus shown in FIG. 2 whichcomprised four plates (600 cm² /plate) and was dispensed evenly over allthe plates as illustrated in FIG. 3. The cells were grown at 37° C., onthe plates (in a horizontal position) for 5 days with occasional gassingthrough a filter with a mixture of 3% CO₂ and 97% air for the purpose ofattaining sufficient oxygen and for adjustment of the pH. The celldensity attained was 5×10⁴ /cm². Microscopic examination showed nomorphological differences between the cells grown in the apparatus ofthe present invention and those grown in the conventional Roux bottle.

EXAMPLE 2

A line of cells derived from rabbit kidney, RK₁₃ cells (1.2×10⁸) grownin conventional tissue culture bottles were digested with a mixture of0.02% EDTA and 0.25% trypsin and suspended in a nutrient mediumcomprising:

Eagle's basal medium--1848 mls.

7.5% Sodium bicarbonate--32 mls.

3.0% Glutamine--20 mls.

Calf serum--100 mls.

The cell suspension was poured and dispensed into the apparatus of FIG.6 comprising 10 dishes with a cell attachment area of 1000 cm² /dish.The cell attachment plates were maintained in a horizontal position for5 days with occasional aeration. Microscopic examination with along-focus microscope showed that RK₁₃ cells formed a confluentmonolayer on every cell attachment plate. The old culture medium wasdiscarded and all the cell sheets were exposed to Newcastle diseasevirus (NDV) at an m.o.i. of 20 for inducing interferon. After absorptionof the virus for one hour at 37° C., the non-absorbed NDV was removed bywashing with serum-free medium. The cell sheets were replenished with 2liters of fresh serum-free medium and further incubated at 37° C., for24 hours, after which the culture fluid was collected, made clear bycentrifuging, acidified to a pH of 2 to inactivate the NDV for fourdays, and neutralized. The neutralized preparation was analyzed for itsviral interfering activity using vesicular stomatitis virus as thechallenger virus. An interferon titer of 30,000 international units/ml.was obtained.

EXAMPLE 3

A strain of human diploid cells (2×10⁷ cells) derived from human fetallung, cultivated in four Roux bottles of 150 cm² /bottle was trypsinizedand suspended in a nutrient medium comprising:

Eagle's basal medium--1750 mls.

7.5% Sodium bicarbonate--32 mls.

3.0% glutamine--20 mls.

Calf serum--200 mls.

The cell suspension was placed in the reservoir and poured into theapparatus as shown in FIG. 8, having overflow passages and comprising apolycarbonate container and ten glass dishes 20 cm×30 cm in size. Theapparatus was inclined to the degree necessary for 200 mls. of liquid tobe contained on each dish, and then returned to the horizontal position.The cells attached to the submerged bottom of each dish and grew to formconfluent monolayers on each dish after 5 days of cultivation at 37° C.The old medium was removed and the sheets were replenished with a newmedium comprising:

Eagle's basal medium--1910 mls.

7.5% Sodium bicarbonate--32 mls.

3.0% Glutamine--20 ms.

Calf serum--40 mls.

After cultivation at 37° C. for one day, the medium was again removed.Five hundred milliliters of solution of a potent interferon inducer,polyriboinosinic polyribocytidylic acid complex (Poly IC, 10 μg./ml.)were added and dispensed evenly (50 mls. each) to all the cell sheetsand remained in contact with the cell sheets for one hour at 37° C.After removal of non-absorbed poly IC, the front panel of the containerwas detached and each dish sequentially removed and irradiated withultraviolet light (70,000 erg/cm²) under aseptic conditions. Theirradiated dishes were immediately returned to the container and theapparatus reconstructed. Serum-free medium (2 liters) was then fed ontothe poly IC-stimulated and UV-irradiated cell sheets on each dish.Incubation was continued for an additional 24 hours at 37° C., afterwhich time the supernatant fluid (about 2 liters) was harvested andfound to contain 10,000 international units/ml. of human interferon.

Changes and modifications of the apparatus and method of this inventionmay be made within the scope of the appended claims.

What is claimed is:
 1. Apparatus for propagating tissue culture cells ina static culture system comprising:a vessel reservoir for liquid culturemedium, said vessel having four vertical walls at opposite ends of afirst horizontal dimension thereof, a plurality of substantiallyparallel spaced apart plates disposed within said vessel for providingsurfaces to which cell growth can attach and liquid and gas phases canexist, said plates being enclosed and in sealing relationship with saidvertical walls along three walls to form individual dishes, the portionof the plates along the wall not in sealing relationship with the wallbeing raised so that when the apparatus is positioned with said platesin a horizontal position all raised portions stand in a substantiallyvertical position and the liquid culture medium is maintained on saidindividual dishes, whereby said apparatus has at least one inter-dishpassage, and at least one inlet and outlet means on said apparatus forpassage of medium and gas.